DO ANTHOCYANINS FUNCTION AS OSMOREULATORS IN LEAF TISSUES? Linda Chalker-Scott Division of Ecosystem Sciences, College of Forest Resources Box 354115, University of Washington, Seattle, WA 98195 Phone: (206) 685-2595 Fax: (206) 685-2692 Email: [email protected] 1 Keywords: anoxia, anthocyanins, avoidance, cold, drought, freezing, hardiness, mineral, nutrient, osmotic, ozone, resistance, salt, stress, sugar, tolerance, water 2 ABSTRACT Water stress can be induced in plant tissues, directly or indirectly, by a number of environmental conditions. Plants exposed to drought, heat, cold, wind, flooding, or saline conditions often synthesize foliar anthocyanins in response. Although previously thought to function as a UV screen, anthocyanins may instead serve to decrease leaf osmotic potential. The resulting depression of leaf water potential could increase water uptake and/or reduce transpirational losses. Combined with other anti-stress activities attributed to anthocyanins (including their solar shield and antioxidative capacities), this phenomenon may allow anthocyanin-containing leaves to tolerate suboptimal water levels. The often transitory nature of foliar anthocyanin accumulation may allow plants to respond quickly and temporarily to environmental variability rather than through more permanent anatomical or morphological modifications. 3 I. Introduction Generally associated with the brilliant red, blue, and purple coloration seen in floral tissues, anthocyanins (and the closely related betacyanins) are also common foliar constituents in a diverse assortment of plants. Anthocyanins are water- soluble pigments derived from flavonoids via the shikimic acid pathway. While foliar anthocyanins may be permanent leaf components in some species, they are developmentally or environmentally transient in many others. Perhaps best known are the transitory autumnal anthocyanins associated with deciduous leaf senescence in temperate regions. At the other end of the developmental spectrum are the juvenile anthocyanins, which appear at bud break and generally disappear after leaves are fully expanded. Finally, anthocyanins can be environmentally transient, appearing and disappearing with changes in photoperiod, temperature, and other signals. The relationship between transient anthocyanins and environmental stress resistance has been recently reviewed (Chalker-Scott, 1999). Regardless of the developmental or environmental signal responsible, anthocyanic transcience can represent a significant metabolic cost to the plant. Energy is required to modify flavononal precursors and form anthocyanins de novo; likewise, their degradation also requires energy. A second potential cost of anthocyanin accumulation is the resulting interference with the light reactions of photosynthesis. Because of their ability to absorb blue and reflect red 4 wavelengths, anthocyanins in the upper epidermis or mesophyll of leaves theoretically compete with light harvesting by chlorophyll and carotenoids. Reductions in photosynthetic rates have been noted in red-leafed varieties of Coleus (Burger and Edwards, 1996) and Capsicum annuum (Bahler et al., 1991), spring flushing leaves of Brachystegia spp. (Choinski and Johnson, 1993, Tuohy and Choinski Jr., 1990), and the red juvenile leaves of several rainforest tree species (Woodall et al., 1998). The competitive advantage afforded to these plants by accumulating anthocyanins must outweigh the costs associated with manufacturing and storing these compounds. This may especially be true under environmentally stressful conditions. In this chapter the relationships between anthocyanins and abiotic environmental stressors will be explored at the whole plant level, particularly those stressors that directly or indirectly induce dehydration stress. These stressors include drought, osmotic disrupters, cold temperature, and anoxia. Evidence in the literature that coincidentally or deliberately links the presence of anthocyanins with induced resistance will be discussed. This concept of environmental resistance can then be applied to specific life history events in an attempt to explain why some anthocyanins are developmentally transient in the context of osmotic regulation. 5 II. Interactions Among Environmental Stressors, Plant Water Content, and Anthocyanins A. Drought Stress The term “drought” is generally used in reference to an abnormal reduction in available water. Certain environments characterized as arid, therefore, are not droughty unless they receive a significantly less than normal amount of rainfall. For the purposes of this chapter, the phrase “drought stress” will be used more generally to refer to any condition in which water may be a limiting factor. 1. Impact of Drought Stress upon Plant Water Relations Lack of available water, whether or not a normal occurrence in a given environment, has the immediate effect of decreasing turgor, most especially in the leaves. As evapotranspiration exceeds water uptake, loss of turgor generally triggers stomatal closure and a decrease in photosynthetic activity. Under prolonged stress, cellular dehydration and eventually plasmolysis will occur in drought-sensitive species. Plants that survive drought conditions generally have small, thick leaves with substantial cuticles, pubescence and/or other structural modifications (Curtis et al., 1996; Li et al., 1996). In contrast, actively growing tissues, whose cell walls are still expanding, often increase vacuolar solutes to decrease leaf osmotic potential, allowing the plant to remain turgid under low soil water potential conditions. 6 Though most water stress probably occurs as a result of low soil water availability, other environmental variables can induce water stress at the leaf level. Anything that causes a loss of integrity of the leaf cuticle and/or epidermis will induce increased evapotranspiration. Strong oxidizing agents, such as ozone, are able to breach these barriers and have been associated in the literature with leaf water stress (Gunthardt-Goerg et al., 1993; Johnson et al., 1995; Landolt et al., 1994; Maier-Maercker, 1997; Sakata, 1996). Under such conditions, evapotranspiration exceeds water uptake and leaf water stress results. 2. Evidence of Anthocyanin-Mediated Drought Resistance a. Linkages to Morphological Adaptations to Drought Both anecdotal and experimental observations have linked drought tolerance with foliar morphological characteristics such as thickened cell walls and/or cuticles, increased pubescence, and anthocyanin accumulation. Several decades ago, research on Populus spp. revealed positive correlations between petiole hairiness and anthocyanin content (Fritzsche and Kemmer, 1959; Kemmer and Fritzsche, 1961). Even earlier work on Fraxinus americana populations had determined that the southernmost ecotype exhibited both increased anthocyanin content and leaf pubescence (Wright 1944); presumably, this ecotype would be exposed to hotter, drier conditions than more northern populations. Foliar anthocyanins have 7 also been correlated with pubescence in Fragaria spp. (Sjulin et al., 2000) and glaucousness of Eucalyptus urnigera (Thomas and Barber, 1974). There is evidence that these adaptations to drought are more than coincidental. Recent molecular work with Arabidopsis mutants has linked these characteristics at the genetic level. Shirley et al. (1995) studied several loci responsible for foliar anthocyanin development and discovered that one of these was also responsible for trichome development. Thickened leaves and anthocyanin increases were found in an Arabidopsis mutant adapted to high light conditions (Iida et al., 2000); high light levels are known to cause secondary drought stress. Finally, a population of Arabidopsis mutants with reduced cuticular waxes was found to accumulate more anthocyanins than normal (Millar et al., 1998), presumably as a means to maintain foliar water. b. Linkages to Ecophysiological Adaptations to Drought Although not commonly mentioned in context with plants from arid environments, anthocyanins are nevertheless associated with water stress in a variety of studies. Perhaps the best example of anthocyanin-associated drought tolerance is that exhibited by resurrection plants. These plants, which include the genera Craterostigma, Myrothamnus, and Xerophyta, are able to lose most of their available water, yet remain viable for extended periods of time. During this 8 process, resurrection plants replace their large central vacuole with smaller vacuoles and increase their anthocyanin content three- to fourfold over normal levels (Farrant, 2000; Sherwin & Farrant, 1998). Less dramatic but nonetheless effective examples of drought-induced anthocyanin accumulation are found in the literature. Increases in anthocyanins and/or associated flavonoids have been noted in drought-stressed Cotinus spp. (Oren- Shamir, personal communication), Cucumis sativus (Zhi-Min et al., 2000), Vaccinium myrtillus (Laura Jaakola, personal communication), Pisum (Allen et al., 1999; Balakumar et al., 1993), Pinus taeda (Heikkenen et al., 1986), Malus cv. ‘Braeburn’ (Mills et al. 1994), Populus spp. (Wettstein-Westersheim and Minelli, 1962), and Quercus spp. (Spyropoulos & Mavormmatis, 1978). These latter authors studied Quercus native to environments with differing levels of water availability: Q. ilex is found in wet evergreen sclerophyllous forests; Q. robur lives in typical mesophytic environments, while Q. coccifera is indigenous to the hottest, driest ecosystems. While all three